This invention relates to novel energy transfer conjugates (ETC's) or labeling agents comprising a chemiluminescent acridinium or benzacridinium derivative covalently attached with a luminophore. This invention also relates to a class of ETC's with ranges of emission wavelength which are distinctively different from or minimally overlapped with those of the chemiluminescent acridinium or benzacridinium derivatives. This invention further relates to the novel application of ETC as a chemiluminescent label in the determination of analytes in a sample, such as diagnostic tests.
Since the development of the polysubstituted aryl acridinium ester (DMAE in U.S. Pat. No. 4,745,181) and its derivatives such as the 3-Methoxy-substituted DMAE, benz[b]acridinium ester (LEAE in U.S. Pat. No. 5,395,752), and 2-Methoxybenz[b]acridinium ester with emission wavelength maxima which are 6 nm shorter or, 96 nm and 122 nm longer than that of the parent DMAE (emission maximum of 428 nm), we continue our research to identify novel acridinium compounds and their synthesis, particularly those that extend the emission wavelength of acridinium compounds including specifically DMAE and generally its well known analogs, e.g., the relatively stable acridinium esters with mono-ortho substituted phenoxy moiety (EP 0609885A1; M. Kawaguichi et al., "Stabilized Phenyl Acridinium Esters For Chemiluminescent Immunoassay--Bioluminescence and Chemiluminescence, Proceedings of 9th International Symposium 1996", Edited by Hastings, Kricka and Stanley, John Wiley & Sons, 1997, pp. 480-484), and the acridinium sulfonylamides (U.S. Pat. No. 5,468,646). The ultimate purpose of the research is to a) improve or eliminate the minimal emission spectral overlap between DMAE and LEAE, b) discover another chemiluminescent label for biomolecules which has emission wavelength longer than that of LEAE and is minimally overlapped with the latter, c) increase the quantum yield of DMAE. Any efforts directed toward these goals must proceed under the pre-requisite that the new chemiluminescent labels will emit light under the same triggering conditions. Accomplishment of the objective (a) would eliminate the need of applying the cross-talk correction routine in the dual-analyte binding assays that employ the dual labels of DMAE and LEAE. With the achievement of objective (b), a third chemiluminescent label would be available for the development of a triple-analyte binding assays. Objective (c) represents one of the possible approaches which is based on the rationale that longer emission chemiluminescent compounds will have lower energy state, hence the probability of populating the chemically-converted, excited state species will be improved to allow better over-all quantum yield.
For the design of acridinium derivatives capable of emitting light at any desired spectral ranges, the present invention adopted the well-known general concept of energy transfer. Unlike the many reported instances of intermolecular energy transfer that involves two separate donor and acceptor molecules, where the donor can be a chemiluminescent compound or a luminophore, and the acceptor is always a luminophore, we have our novel design of linking the acridinium compound and the acceptor luminophore together into one molecule in order to achieve more efficient intramolecular energy transfer. In doing so, we can effectively channel the chemical energy generated in the acridinium moiety to the selected luminophore moiety, so that the latter can be excited and emit light at its characteristic and expected wavelength range. With regard to the design goals of operating uniquely within the system of chemiluminescent acridinium compounds and the requirements for high sensitivity (femtomolar, fM) in aqueous media, fast-production (in a few seconds) of light, monophasic emission (meaning only one emission maximum), and longer emission maxima, some distinct differences exist between inter- and intramolecular energy transfer phenomena and should be mentioned as the following:
M. M. Rauhut et.al. [J. Amer. Chem. Soc., 89, 6515 (1967)] first reported intermolecular energy transfer as observed in the chemiluminescence generated from the reactions of electronegatively substituted aryl oxalates with hydrogen peroxide and fluorescent compounds. Since in general, the effectiveness of energy transfer diminishes at the rate of the sixth power of the distance between the donor and acceptor, [in other words the donor and acceptor molecules should be within the distance of &lt;10 nm to give 20-100% efficiency of transfer; see Clin. Chem., 29 (9), 1604 (1983) and Ann. Rev. Biochem., 47, 819 (1978)], millimolar (mM, 10.sup.12 folds higher than the requirement) concentration of the acceptor is required. Furthermore there are several other drawbacks in the chemiluminescent peroxalate system which makes it unsuitable for use in biological assays. These drawbacks include long duration (&gt;one minute) for the total light emission, decreased stability of the oxalate in aqueous media, and the need of an organic solvent to solubilize the fluorophore.
Similarly J. Hadjianestis [J. Photochem. Photobiol., A: Chem, 69, 337 (1992)] reported less than quantitative (79%) energy transfer between chemiluminescent luminol and fluorescein. To achieve the maximal result, both donor and acceptor are required to reach only at micromolar (uM, 10.sup.9 folds higher than the requirement) concentration, due to the concentrating effect of a surfactant, CTAC. However, the biphasic profile of the emission spectra generated from this luminol/fluorescein system extending broadly from 350-600 nm renders it unsuitable for a highly sensitive, multi-analyte binding assay.
Other observations of intermolecular energy transfer that have been reported include cis-diethoxy-1,2-dioxetane to perylene [T. Wilson, et.al., J. Amer. Chem. Soc., 93 (17) 4126 (1971)], N-methylacridone to lucigenin [A. E. Mantaka-Marketou, et.al., J. Photochem. Photobiol., A: Chem., 48, 337 (1989); A. Larena, et.al., Monatshefte Chemie, 122, 697 (1991); K. Papadopoulos, et.al., J. Photochem. Photobiol., A: Chem., 75, 91 (1993)]. Aside from the need of high concentrations (uM to mM) of the acceptor, the articles focused on the elucidation of energy transfer and mechanistic studies in the chemiluminescence of dioxetane and lucigenin systems. No application of the energy transfer phenomenon to high sensitivity binding assays was suggested.
The application of intermolecular energy transfer phenomena to immunoassays was first reported by A. Patel, et.al. [Clin. Chem., 29 (9), 1604 (1983)]. A homogenous type immunoassay was developed by utilizing the specific binding property of a hapten conjugated with chemiluminescent isoluminol derivative (ABEI) and an antibody labeled with fluorescein. Since each reagent was added to the assay mix at nanomolar (nM) concentrations which are marginal for intermolecular energy transfer to be observed between the isoluminol and fluorescein moieties, only through the mediation of a specific complex formed between the hapten conjugate and the antibody conjugate that the donor/acceptor molecules have the chance to be pulled into close proximity to allow energy transfer to occur. This immunoassay method is unique in this regard. However, this method suffers from principal drawbacks of limited usefulness, low assay sensitivity (&gt;10.sup.10 analyte molecules/test) inherent in a homogenous assay format and the interference of high background signal originating from the energy donor due to the incomplete energy transfer. Besides, the accuracy of the analyte determination has to depend on the signal ratios taken from the diminishing donor signal and increasing acceptor signal of the assay mix that have significant spectral overlap.
L. E. Morrison, et. al. (EP Patent Application #0070686 A2) teach an enhanced (enzymatic) luminescence immunochemical principle for detecting antigens with multiple binding sites, by employing intermolecular energy transfer from a luminol substrate to an antibody-conjugated fluorophore. This rather complex assay architecture contains also catalase and an antibody-conjugated glucose oxidase as the necessary reagents. The antigen-bound antibody conjugates work together in close proximity to produce specific signal by utilizing the cofactors generated (H.sub.2 O.sub.2) or present (luminol) nearby and providing with the required energy acceptor. The catalase serves as a scavenger for the portion of H.sub.2 O.sub.2 which diffuses away from the antigen/antibody complex, thus minimizing the background chemiluminescence produced by luminol in solution, which is too far to be effectively transferred to the fluorophore. No assay examples were provided to prove the concept is functional.
Minister van Welzijn (NL Patent application #8703075A) described 10-carboxyalkyl-acridinium ester derivatives and suggested its conjugation to an antibody or antigen for use in a homogeneous assay, based on the principle of chemiluminescence intermolecular energy transfer as described by Patel. No example of a functional assay was given.
In the field of intramolecular energy transfer, related prior art can be identified and distinguished from the following:
Several related articles were published concerning intramolecular energy transfer conjugates between chemiluminescent luminol or benzluminol to four other fluorophores, i.e. diphenylanthracene, benzcarbazole, acridone, and benzacridone [E. H. White, et.al., J. Amer. Chem. Soc., 89, 3944 (1967); E. H. White et.al., Mol. Lumin. Int. Conf., 479 (1969), Ed.: E. Lim, Publisher: W. A. Benjamin Inc., New York.; D. F. Roswell, et.al., J. Amer. Chem. Soc., 92, 4855 (1970); D. R. Roberts, et.al., J. Amer. Chem. Soc., 92, 4861 (1970); M. A. Ribi, et.al., Tetrahedron, 28, 481 (1972)]. The quantum yields of such conjugates in aqueous media are all significantly lower than the parent luminol ranging from 26%, 4.4%, 8%, and about 13% of luminol quantum yield, respectively. No application of these luminol derivatives in diagnostic tests was suggested.
Schaap et.al. (WO #90/07511) described another intramolecular energy transfer system involving the conjugation of adamantanyl dioxetanes with fluorophores, wherein the dioxetane moiety is substituted with a cleavable group X (e.g. phosphate) and upon the leaving of X, which can be triggered enzymatically or chemically, enhanced chemiluminescence evolves. It was reported that the original light emitting species, methyl 3-hydroxybenzoate (MHB) which splits from the native adamantanyl dioxetane during the chemiluminescence process, is inherently a very poor light emitter in aqueous media. By tethering a fluorophore to MHB the excitation energy of MHB can be transferred to the fluorophore which has much better light emission in aqueous media and results in an absolute quantum yield improvement from 0.017% to about 1-2% in the presence of CTAB surfactant. The uses of this fluorophore-tethered stable dioxetane were not clearly taught nor claimed in the application. Moreover, its usefulness in enzyme-linked immunoassays and enzyme-linked DNA probes as well as direct, chemically triggerable labels for biomolecules was only briefly alluded in the Field of Invention section. As in the earlier related patent application on other stable dioxetanes by Bronstein (WO 88/00695), the fluorophore-tethered stable dioxetane of Schaap will most likely find its use as a substrate for enzyme-linked tracers or probes in binding assays to achieve enhanced chemiluminescence. For this type of use, however, Schaap did not provide any teaching as to how a multianalyte assay system can be devised, and it certainly would not be possible, unless different enzyme-substrate systems that are non mutually interacting have been made available and clearly demonstrated. Furthermore, the suggestion for an alternative use of this fluorophore-tethered stable dioxetane as a direct label for biomolecules also lacks supportive evidence because in the specification and claim of the general structure of fluorophore-tethered dioxetane, one can not find provisions for specific functional group that would make its direct labeling of the biomolecules possible. The suggestion can be meaningful only if such biomolecule conjugate can be prepared and its stability demonstrated. Additionally, although the well known stable dioxetane with cleavable group X is a very useful means for signal amplification when serving as a substrate for enzyme-linked tracer, it suffers the drawback of slow emission of light due to not only the required long lag time (20 min or more) in the enzymatic cleavage of X, but also the slow decay process (t.sub.1/2 greater than one min) that begins with the cleaving of X and ends up with the light emission.